We started studying the structures of yeast prions in 1998, in collaboration with R Wickner (NIDDK), focussing initially on Ure2p, a negative regulator of nitrogen catabolism. We showed that its N-terminal domain is responsible for prionogenesis, while the C-terminal domain which performs its regulatory function remains folded in filaments but is inactivated by a steric mechanism. In our amyloid backbone concept, the prion domains form the filament backbone and are surrounded by the C-terminal domains. In 2005, we published the parallel superpleated beta-structure model for the amyloid backbone. It envisages arrays of parallel beta-sheets generated by stacking monomers with planar beta-serpentine folds. Topologically similar structures are good candidates for certain other amyloid fibrils, including amylin and growing support for models of this kind is appearing in the scientific literature. Ongoing work is aimed at testing and refining this model; exploring its range of applicability; and investigating fibril polymorphism. In FY08, we focussed on two areas: [unreadable] [unreadable] (1) Fibrillation of the prion domain of the HET-s prion of the filamentous fungus, Podospora anserina. This prion differs from the yeast prions, Ure2p and Sup35p, in being a gain-of-function prion, and in not having an abnormally high concentration of Asn/Gln residues. In 2007, we published a paper showing by electron diffraction that Het-s fibrils have a cross-beta structure (as anticipated), and by scanning transmission electron microscopy that it has an axial packing density of 1 subunit per 0.94 nm - half the density of Ure2p and Sup35p fibrils, and in agreement with a published model, the stacked beta-solenoid. It follows that the respective amyloid architectures are basically different. In another study, also completed during 2007, we compared the amyloids formed by the HET-s prion domain in vitro at different pH's. Fibrils formed at pH 7 from those formed at pH 2 on morphological grounds, in having lower specific infectivity, and in other properties such as ThT-binding. The altered properties of amyloid fibrils assembled at pH 2 may arise from a perturbation in the subunit fold or in fibrillar stacking. The relatively homogeneous fibril structures formed by HET-s at pH's 3 - 4 commend it as a potential specimen for cryo-EM analysis. For this purpose the expression system for HET-s prion domain in E. coli was first optimized, eventually achieving very high yields (>100 mg from 1 L of liquid culture). In EM studies to date, we have detected a previously unsuspected long-range repeat in singlet fibrils as well as a short-range repeat that is longer than the canonical 0.94 nm. Analysis of these data is ongoing.[unreadable] [unreadable] (2) Residues 1-89 constitute the full-length prion domain of the Ure2p protein and these polypeptides form the amyloid backbone of the URE3 prion. We collaborated with R Tycko (NIDDK), an expert in solid-state NMR spectroscopy to use that technique to probe the structure of amyloid fibrils formed by Ure2p1-89. The resulting data revealed that the substructure of the fibril is based on a parallel structure for adjacent beta-strands, with at most a one-residue shift from exact in-register alignment. An in-register parallel beta-sheet structure is consistent with the parallel super-pleated beta-structure model that we proposed in 2004. This arrangement permits polar zipper interactions among sidechains of Gln and Asn residues and explains the tolerance of URE3 to scrambling of the sequence. Two-dimensional NMR spectra of uniformly labeled fibrils, even in a fully[unreadable] hydrated state, show linewidths that greatly exceed those observed (by others) in solid state NMR measurements on HET-s prion domain fibrils, indicating a lower degree of structural order in the Ure2p fibrils. The observed high degree ofl order in HET-s fibrils as detected by solid state NMR data is therefore not a universal characteristiic of prion proteins.